High-throughput glutathione s-transferase polymorphic allele assay design

ABSTRACT

A high-throughput assay for characterizing a subject&#39;s genetic makeup is disclosed. Specifically a high-throughput assay utilizing PCR is disclosed that permits the rapid and accurate characterization of a subject&#39;s inherited alleles of the polymorphic glutathione S-transferase (GST) genes GSTM1, GSTM3, GSTP1, and GSTT1. This method allows detection of the specific alleles inherited, including the gene dosage of GSTM1 and GSTT1 while not requiring restriction endonuclease digestion of the PCR products in order to detect length differences. Further, the method allows all analyses to be performed simultaneously in the same gel lane, thus further adding efficiency and cost-effectiveness.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to high-throughput assays forcharacterizing a subject's genetic makeup. Specifically, the instantinvention is a high-throughput assay that permits the rapid and accuratecharacterization of a subject's inherited alleles of the polymorphicglutathione S-transferase (GST) genes GSTM1, GSTM3, GSTP1, and GSTT1.

[0003] 2. Background of the Invention

[0004] Recent cancer research has shown that the presence of variouspolymorphisms of glutathione S-transferase (“GST”) correlates withaltered risk for certain cancers and altered response and toxicity fromcurrently known and used cancer treatments, including chemotherapy. TheGST family of enzymes has been shown to function in the detoxificationof a broad range of environmental and non-environmental DNA-damagingcarcinogens such as polyaromatic hydrocarbons like those found in first-and second-hand cigarette smoke. Additionally, however, these enzymesare capable of detoxifying chemotherapeutic compounds such as alkylatingagents and anthracyclines as well as reactive oxygen species andperoxides. See S. Tsuchida and K. Sato, Critical Reviews in Biochemistryand Molecular Biology, 27(4, 5):337-384 (1992), and S. A. Weitzman andL. I. Gordon, Blood, 76(4):655-663 (1990).

[0005] This family of enzymes has been subdivided into four subclasses,including GSTM1, GSTM3, GSTP1, and GSTT1. Ali-Osman et al., J. Biol.Chem., 272(15):10004-10012 (1997); Fryer et al., Biochem. J.,295:313-315 (1993); Inskip et al., Biochem. J., 312:713-716 (1995); andPemble et al., Biochem. J., 300:271-276 (1995). A first of these is theGSTM1 class, which includes the following allelic variants: GSTM1null,GSTM1*A, and GSTM1*B. Fryer et al., Biochem. J., 295:313-315 (1993).

[0006] GSTM1*null is thought to result from an unequal crossing-over ata duplicated region between the GSTM1 and the GSTM2 loci. Pearson etal., Am. J. Hum. Genet., 53:220-233 (1993); and Xu et al., J. Biol.Chem., 273:3517-3527 (1998). As with other null alleles, this oneproduces no functional product and thus acts as a recessive gene.GSTM1*A and GSTM1*B are polymorphic alleles commonly thought to resultfrom a C to G substitution at codon 173. This change results in a changefrom the Lys¹⁷³ of GSTM1*A to the Asn¹⁷³ of GSTM1*B. This change altersa hinge region between alpha helices which is involved in GSTM1dimerization.

[0007] A second subgroup of GST enzymes is dubbed the GSTM3 class,including the GSTM3*A and GSTM3*B allelic variants. These alleles arethought to result from a 3 base-pair deletion in intron 6 whichgenerates a YY1 negative transcription factor recognition site inGSTM3*B that does not exist in GSTM3*A.

[0008] A third such subgroup of GST enzymes is the GSTP1 group whichincludes GSTP1*A, GSTP1*B, and GSTP1*C. A GSTP1*D allele has beenobserved, but only in very rare circumstances. These polymorphic allelesresult from A to G and C to T transitions at nucleotides +313 of exon 5and +341 of exon 6, respectively. Specifically, GSTP1*A codon 104 isATC, coding for Ile¹⁰⁴, and codon 113 is GCG, for Ala¹¹³. GSTP1*B codon104 is GTC, coding for Val¹⁰⁴, and codon 113 is GCG for Ala¹¹³. GSTP1*Ccodon 104 is GTC, coding for Val¹⁰⁴, and codon 113 is GTG for Val¹¹³.GSTP1*D codon 104 is ATC, coding for Ile¹⁰⁴, and codon 113 is GTG forVal¹¹³.

[0009] A fourth group of GST enzymes is the GSTT1 subgroup, whichincludes GSTT1*null and GSTT1. As with the other null allele notedabove, the GSTT1*null allele produces no functional product, thusoperating as a recessive allele.

[0010] There are techniques extant in the art for assessing whichalleles are present in an individual's genotype. Most of these assays donot allow investigators to determine gene dosages. Here, the term “genedosage” is used to denote whether one or both alleles were present whena PCR product suggested the presence of at least one non-null allele.Further, most of the currently used assays do not differentiate betweenthe non-null GSTM1 alleles GSTM1*A and GSTM1*B. These methods alsogenerally require that the PCR products undergo restriction endonucleasedigestion to allow the determination of genotypes, thus adding extracomplexity and expense to the method.

[0011] Kristensen et al. reported one such assay in 1998. Kristensen etal., Pharmacogenetics, 8:441447 (1998). This assay was able to evaluateonly the polymorphisms of GSTM1, GSTP1, and GSTT1, while not beingcapable of distinguishing between GSTM1*A and GSTM1*B. Further, whenlooking at GSTP1, the assay examined only codon 104, ignoring thepolymorphisms showing changes at codon 113. Finally, as with other knownassays, this assay method did not examine the gene dosage of eitherGSTM1 or GSTT1.

[0012] The availability of simple, effective assays could allow theanalysis of individuals' genomes in order to detect risk for specificdiseases and cancers and to allow the development of individualizedprevention and/or treatment strategies. Further, in regard to thespecific family of enzymes referenced above, specific, accurate assayscould allow the development of tailored therapeutic regimens forpatients predicted to have decreased therapeutic response to medicaltherapy, including cancer therapy, based on their expression of GSTenzymes, or for patients predicted to have increased therapy-relatedtoxicity. Additionally, such assays would simplify the implementation ofpatient-specific utilization of allele-specific small-moleculeinhibitors for the purpose of reversing chemotherapy resistance amongcancers, such as those over-expressing certain GST polymorphic alleles.

[0013] From the above, it is apparent that it would be an improvement inthe art to provide a high-throughput assay method for rapidly,inexpensively, and accurately characterizing the GST alleles present ina subject. It would be a further advancement in the art to provide ahigh-throughput GST assay method which is capable of accuratelydetermining the gene dosage of GSTM1 and GSTT1 using competitive PCR.Additionally, it would be a further advancement in the art to providesuch an assay design which is simpler, faster, and cheaper than thosecurrently known in the art because it does not require restrictionendonuclease digestion of PCR products in order to elucidate the lengthdifferences between GST alleles. Similarly, it would be an improvementin the art to allow the assay of all of the PCR products simultaneouslyin a single gel lane, which would yield further savings in time andexpense. Finally, it would be an improvement in the art to provide ahigh-throughput assay method that would comprehensively assay all fourGST polymorphs and their alleles, including null alleles. Such an assaymethod is disclosed herein.

SUMMARY OF THE INVENTION

[0014] The present invention provides methods and apparatus fordetecting the presence of glutathione S-transferase alleles using PCRmethods and unique primers in such a way as to allow accurate genotypingby detecting the alleles of GSTM1, GSTM3, GSTT1, and GSTP1 present in asample, while also allowing the detection of the gene dosage of GSTM1and GSTT1.

[0015] This invention is important to the feasibility and future successof large population studies of how differences in the genotype of thealleles of glutathione S-transferase correlate with risk for cancer, aswell as how the alleles correlate with possibility of risk andsuccessful outcomes of therapy for a group of diseases including cancer.Specifically, GST enzyme polymorphisms have been correlated with risksfor cancer and with risks of altered response and toxicity from cancertreatments. Existing assays for the alleles of GSTM1, GSTM3, GSTT1, andGSTP1 require multiple PCR runs followed by endonuclease digestion ofthe PCR products in order to detect the length differences between theGST polymorphic alleles. Most currently known and practiced assays areincapable of demonstrating the presence of the alleles of all fourpolymorphs. Further, both GSTM1 and GSTT1 have null alleles, andexisting assays cannot show the presence of these alleles or thecorresponding gene dosage of GSTM1 and GSTT1. Finally, current analysismethods are very expensive. This fact places strong restrictive limitson the size of the population evaluated in many recent studies.

[0016] The instant invention overcomes these limitations by usingcarefully designed PCR primers in paired PCR competitions to allow eachof the alleles of the polymorphisms of GST to be detected, includingboth null alleles. Table 1 shows one set of possible primers that can beused with the instant invention. Forward primers are indicated by “Fwd,”and reverse primers are indicated by “Rvs.” TET and FAM are fluorescenttags that can be used to detect sequences by automated polyacrlamide gelelectrophoresis. Boxed sequences indicate non-sequence specific tailsused to create PCR product length polymorphisms. Underlined nucleotidesindicate single nucleotide polymorphisms. TABLE 1 High ThroughputGenotyping: PCR Primers Primer Fwd/ Gene name Rvs Primer Sequence PCRProduct GSTM1 M1F-A Fwd 5′-(TET)TTGGGAAGGCG 142 bp (GSTM1*A)TCCAAGCAC-3′ M1F-B Fwd

145 bp (GSTM1*B) M1R Rvs

 0 bp (GSTM1*null) GSTM3 M3F Fwd

287 bp (GSTM3*A) M3R Rvs

284 bp (GSTM3*B) GSTP1 P1-10FA Fwd

150 bp (Ile¹⁰⁴) P1-104FG Fwd

153 bp (Val¹⁰⁴) P1-104R Rvs

P1-113FC Fwd

130 bp (Ala¹¹³) P1-113FT Fwd 5′-(FAM)GGTGTCTGGCA 126 bp(Val¹¹³)GGAGGC-3′ P1-113R Rvs

GSTT1 T1F Fwd 5′-(FAM)TTCCTTACTGGT 255 bp (GSTT1) CCTCACATCTC-3′ T1R Rvs

 0 bp (GSTT1*null)

[0017] In one embodiment, the invention is a method including the stepsof obtaining a sample of genomic DNA and conducting three separate PCRruns of portions of the sample. After this, the alleles may be detected.In one embodiment of the invention, a first portion of the sample isamplified by PCR with primers for GSTM1 and CDK2; a second portion isamplified with primers for GSTT1 and GSTM3; and a third portion of thesample is amplified with primers for GSTP1. Following this, the allelesmay be detected. In the above method, at least one of the primers usedfurther has a fluorescent such as TET, FAM(6FAM), 5FAM, TAMRA, HEX,R110, JOE, RG6, NED, ROX (Applied Biosystems, Foster City, Calif.).Further, the PCR products of the amplification of the first, second, andthird portions of the sample are combined prior to the step of detectingthe presence and the gene dosage of the alleles. This detection step maybe accomplished by conducting a gel electrophoresis of the sample usinga single lane. This allows cost savings over traditional methods. Thedetection step may next involve fluorescence detection to determine thepresence of PCR products for the specific alleles. In addition, thedetection step may further involve comparing the areas under the peak ofGSTM1*A or GSTM1*B PCR products with the area under the peak of the CDK2PCR products to determine the gene dosage of GSTM1; as well as comparingthe areas under the peak of the GSTM3*A or GSTM3*B PCR products with thearea under the peak of the GSTT1 PCR products in order to determine thegene dosage of GSTT1.

[0018] The detection step noted above may be varied significantly withinthe scope of the instant invention. In some forms of the invention,capillary electrophoresis is used in the place of typical gelelectrophoresis, followed by fluorescence detection to determine thepresence of PCR products for the specific alleles and comparison of theareas under the peak of the GSTM1*A or GSTM1*B PCR products with thearea under the peak of the CDK2 PCR products to determine the genedosage of GSTM1 and comparison of the areas under the peak of theGSTM3*A or GSTM3*B PCR products with the area under the peak of theGSTT1 PCR products to determine the gene dosage of GSTT1.

[0019] In other forms of the method of the instant invention, theprimers include internally-biotinylated nucleotides. In these methods,the step of detecting the presence and gene dosage of the allelescomprises the steps of combining the PCR products of the amplificationof the first, second, and third portions of the sample; exposing themixture to streptavidin-coated magnetic or non-magnetic beads; anddetecting the presence or absence of allele-specific PCR productsthrough the use of a microplate reader capable of indicating thepresence or absence of allele-specific PCR products.

[0020] Other forms of the invention utilize real-time PCR to amplify anddetect the alleles of GSTM1, GSTM3, GSTT1, and GSTP1. This method isuseful due to its relative ease and simplicity and its ability toamplify and detect the alleles and determine the gene dosages of GSTM1and GSTT1 alleles all at the same time and without using specializedequipment, excepting the real time PCR machine itself.

[0021] In other forms, the method includes primers that include at leastone radiolabeled nucleotide and uses methods known in the art fordetecting the radiolabeled molecules, including electrophoresis followedby autoradiography or phosphoimaging and related techniques.

[0022] In other embodiments of the invention, it is a high-throughputassay for the detection of glutathione S-transferase polymorphic allelescomprising the steps of: obtaining a sample of genomic DNA, conductingPCR amplification of the genomic DNA using primers for GSTM1, CDK2,GSTT1, GSTM3, and GSTP1; combining the PCR reaction products into onemixture; and detecting the presence and the gene dosage of the alleles.Here, the primers may further include labels such as: fluorescentnucleotide dyes (TET, FAM(6FAM), 5FAM, TAMRA, HEX, R110, JOE, RG6, NED,ROX)(Applied Biosystems, Foster City, Calif.), biotinylateddeoxynucleotides, radioactive phosphorus deoxynucleotides, orradioactive sulfur phosporothioates, and others that could functionproperly in this application. The detection step also involves loadingthe mixture into a single gel lane and conducting gel electrophoresis orcapillary electrophoresis.

[0023] A presently preferred embodiment of the instant invention is amethod of identifying the alleles of glutathione S-transferase presentin a sample of genetic material which includes three distinct PCR stepswhich may be performed in any order. A first step is to conduct the PCRamplification of the GSTM1 alleles and CDK2 present in a first portionof a genetic sample using primers comprising polynucleotide sequencessubstantially identical to SEQ ID NO: 1, nucleotides 3-23 of SEQ ID NO:2, nucleotides 9-31 of SEQ ID NO: 3, SEQ ID NO: 14, and SEQ ID NO: 15;wherein at least one of said primers further comprises a signal marker.A second step is to conduct the PCR amplification of the GSTT1 and GSTM3alleles present in a second portion of the sample using primerscomprising polynucleotide sequences substantially identical tonucleotides 7-26 of SEQ ID NO: 4, nucleotides 7-26 of SEQ ID NO: 5, SEQID NO: 12, and nucleotides 6-27 of SEQ ID NO: 13; wherein at least oneof said primers further comprises a signal marker. A third step is toconduct the PCR amplification of the GSTP1 alleles present in a thirdportion of the sample using primers comprising polynucleotide sequencessubstantially identical to nucleotides 7-25 of SEQ ID NO: 6, nucleotides128 of SEQ ID NO: 7, nucleotides 6-23 of SEQ ID NO: 8, nucleotides 421of SEQ ID NO: 9, SEQ ID NO: 10, and nucleotides 7-25 of SEQ ID NO: 11;wherein at least one of said primers further comprises a signal marker.The detection step follows these amplification steps, and shows thepresence or absence of the alleles of GSTM1, GSTM3, GSTT1, and GSTP1,and the gene dosage of GSTM1 and GSTT1.

[0024] As with other embodiments, the signal marker of those primerswhich have markers such as fluorescent dyes (TET, FAM(6FAM), 5FAM,TAMRA, HEX, R110, JOE, RG6, NED, ROX) (Applied Biosystems, Foster City,Calif.), biotinylated deoxynucleotides, radioactive phosphorusdeoxynucleotides, or radioactive sulfur phosporothioates, and mayinclude other suitable markers. Also, in some of the methods, the PCRproducts of the amplification steps are combined prior to the step ofdetecting the presence of the alleles of GSTM1, GSTM3, GSTT1, and GSTP1,and the gene dosage of GSTM1 and GSTT1. Further, this step may simplycomprise analyzing the allele makeup and gene dosage of the sample usinga single lane of a gel electrophoresis. This step could also bedetecting the presence of the alleles of GSTM1, GSTM3, GSTT1, and GSTP1,and the gene dosage of GSTM1 and GSTT1 using capillary electrophoresis.

[0025] In other forms of the instant invention, the primers mayadditionally comprise internally-biotinylated nucleotides. In these, thestep of detecting the presence and gene dosage of the alleles includesthe steps of combining the PCR products of the amplification of thefirst, second, and third portions of the sample; exposing the resultingmixture to streptavidin-coated magnetic or non-magnetic beads; anddetecting the presence or absence of allele-specific PCR productsthrough the use of a microplate reader capable of indicating thepresence or absence of allele-specific PCR products.

[0026] In still other forms of the invention, the steps of amplifying afirst portion of the sample with primers for GSTM1 and CDK2; amplifyinga second portion of the sample with primers for GSTT1 and GSTM3;amplifying a third portion of the sample with primers for GSTP1; anddetecting the presence and the gene dosage of the alleles areaccomplished using real-time PCR, wherein amplification and detection ofalleles is conducted simultaneously. In some of these embodiments, thesignal markers comprise radiolabeled nucleotides. In these, the step ofdetecting the presence of the alleles of GSTM1, GSTM3, GSTT1, and GSTP1,and the gene dosage of GSTM1 and GSTT1 comprises detecting theradiolabeled PCR products.

[0027] Further, in some embodiments, the instant invention is a novelPCR primer comprising a nucleotide sequence selected from the group ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. In some of these, thePCR primer further includes a signal marker selected such as fluorescentdyes (TET, FAM(6FAM), 5FAM, TEA, HEX, R110, JOE, RG6, NED, ROX)(AppliedBiosystems, Foster City, Calif.). These primers may further compriseradiolabeled nucleotides and/or internally-biotinylated nucleotidesand/or nucleotide analogs within the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWING

[0028] In order that the invention may be more readily understood, amore particular description of the invention briefly described abovewill be rendered by reference to specific embodiments thereof which areillustrated in the appended drawing. Understanding that this drawingdepicts only typical embodiments of the invention and are not thereforeto be considered to be limiting of its scope, the invention will bedescribed and explained with additional specificity and detail throughthe use of the accompanying drawings in which:

[0029]FIG. 1 shows the result of the high-throughput GST polymorphicallele assay of the instant invention for five DNA samples taken fromfive different human cell lines.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Polymorphisms of glutathione S-transferase (GST) enzymes havebeen correlated with clinically significant changes of risk for manydifferent cancers. Specifically, cancer risk differences have beenassociated with polymorphic alleles of GSTM1, GSTM3, GSTP1, and GSTT1.See e.g., Krajinovic et al., Blood, 93:1496-1501 (1999), Jahnke et al.,Am. J. Surg., 172:671-673 (1996), Volm et al., Med. & Ped. Onc.,28:117-126 (1997), and Elexpuru-Camiruaga et al., Canc. Res.,55:4237-4239 (1995). Differences in response, toxicity, and outcome oftreatment for cancer have been associated with polymorphic alleles ofGSTM1 and GSTP1. See e.g., Goto et al., Canc. Res. 56:3725-3730 (1996),and Volm et al., Med. & Ped. Onc., 28:117-126 (1997). These and otherstudies conducted on this topic are retrospective studies, however, andthus prospective examinations of risk and response through nationaltrials should lead to the discovery of better-defined relationshipsbetween the possible GST genotypes and cancer and cancer treatmentsuccess levels.

[0031] GST polymorphic alleles occur at frequencies of at least 2.4-20%,and may be as high as 40-84%. This fact alone renders them socially andmedically important. The frequencies of these alleles vary among diverseethnic groups, as seen in Tables 2, 3, 4, and 5 below. Table 2 containsstatistics regarding the GSTM1 polymorphic allele frequency in diverseethnic groups. The number of patients examined in each ethnic group isdesignated as “n”. GSTM1*null is designated as “null.” GSTM1*A andGSTM1*B are designated as M1*A and M1*B respectively.1 TABLE 2 GSTM1Polymorphic Allele Frequencies among Diverse Ethnic Groups Null: Null:M1*A; Null: M1*B; Ethnic Group (n) null M1*A/M1*B; or M1*A/M1*BMultinational 253 50 50 Washington Co., MD 110 64.5 35.5 (caucasians)African-Americans 59 40.7 59.3 Quebec, Canada 174 64.9 35.1 UnitedKingdom 84 44 66 United Kingdom 300 57.3 42.7 England 113 63.7 56.3Finland 142 43.7 56.3 Berlin, Germany 400 50.7 49.3 Parma, Italy 98 5050 Linxian County, China 45 53 47 Taiwan 150 63.3 36.7

[0032] Table 3 shows GSTM3 polymorphic allele frequencies in twopopulations from Great Britain. The number of patients examined in eachpopulation is designated as “n”. M3*A indicates a GSTM3*A allele, andM3*B indicates a GSTM3*B allele. TABLE 3 GSTM3 Polymorphic AlleleFrequencies M3*A: M3*A: M3*B: Ethnic Group (n) M3*A M3*B M3*A M3*B M3*BGreat Britain 244 84.2 15.8 70.9 25.8 3.4 Great Britain 300 73.7 21.35.0

[0033] Table 4 shows GSTP1 polymorphic allele frequencies among diverseethnic groups from all over the world. The number of patients examinedin each ethnic group is designated as “n”. P1*A, P1*B, and P1*C indicateGSTP1*A GSTP1*B GSTP1*C alleles respectively. TABLE 4 GSTP1 PolymorphicAllele Frequencies among Diverse Ethnic Groups P1*B or Ethnic Group (n)P1*A P1*B P1*C P1*C African-American 112 56.6-58% 39.9% 2.1-5% European199 61.4% 31.6% 2.4% Scottish 155 72.3% 27.7% Norwegian 297 71.2% 28.8%German Caucasian 180 69.5% 30.5% Indian 40 66.4% 24.6% 2.4% Chinese 4980.2% 18.8% 0.2% Linxian County, 36 76.5% 23.5% China Japanese 164 83.5%16.5% Aborigninese 45   89%   11%   0%

[0034] Finally, Table 5 shows GSTT1 polymorphic allele frequencies amongdiverse ethnic groups from all over the world. The number of patientsexamined in each ethnic group is designated as “n”. GSTT1*null isindicated by “null.” “T1” indicates a GSTT1 allele. TABLE 5 GSTT1Polymorphic Allele Frequencies among Diverse Ethnic Groups Ethnic Group(n) Null:null Null:T1 or T1:T1 USA 190 16 84 Washington Co., MD 110 27.272.7 African-American 119 21.8 78.2 African-Americans 59 28.8 71.2Mexican-American 73  9.7 90.3 French Canadian 176 84.1 United Kingdom494 18.4 81.6 Sweden 270  9.6 90.4 Australia 94 19 81 Chinese 45 64.435.6 Linxian County, 45 49 51 China Korean 103 60.2 39.8 Japanese 12644.4 55.6

[0035] The assay design of the instant invention has four advantagesover existing high-throughput assays. Because most GST genotyping assayscurrently known in the art are solely PCR-based, it has not beenpossible for investigators to determine gene dosage in the course of theassay. Gene dosage denotes an evaluation of whether one or both alleleswere present when a PCR product suggested the presence in the genotypeof at least one non-null allele. Further, these assays generally do notdifferentiate between the non-null GSTM1 alleles (GSTM1*A and GSTM1*B).Most of these examinations further require the additional step ofdigesting the PCR products with restriction endonucleases in order todetermine the genotype.

[0036] A first step in the method is to assay for the three polymorphicalleles of GSTM1. The GSTM1 class includes the GSTM1*null, GSTM1*A, andGSTM1*B allelic variants. Fryer et al., Biochem. J., 295:313-315 (1993).GSTM1*null is thought to result from an unequal crossing-over at aduplicated region between the GSTM1 and the GSTM2 loci. Pearson et al.,Am. J. Hum. Genet., 53:220233 (1993); and Xu et al., J. Biol. Chem.,273:3517-3527 (1998). As with other null alleles, this one produces nousable product and thus exists as a functional genetic recessive.GSTM1*A and GSTM1*B are polymorphic alleles commonly thought to resultfrom a C to G substitution at codon 173. This change results in a changefrom the Lys¹⁷³ of GSTM1*A to the Asn⁷¹³ of GSTM1*B. This change altersa hinge region between alpha helices which is involved in GSTM1dimerization. The polymorphic alleles of Glutathione S-0TransferaseGenes are summarized in Table 6. TABLE 6 Polymorphic Alleles ofGlutathione S-Transferase Genes Class/ Allelic Subclass Locus VariantsCodon Significance Mu 1p13.3 GSTM1*null Absent Allele GSTM1 GSTM1*ALys¹⁷³ Hinge between to GSTM1*B Asn¹⁷³ alpha helixes at dimerizationsite GSTM3 1P13.3 GSTM3*A Full Intron 6 Unknown; the GSTM3*B 3bpDeletion deletion generates in Intron 6 a YY1 Negative TransciptionFactor Recognition Site Pi 11q13 GSTP1*A Ile¹⁰⁴(104a), Modified contactsGSTP1 Ala¹¹³(113C) at the binding site GSTP1*B Val¹⁰⁴(104G), forelectrophilic Ala¹¹³(113C) carcinogens (H- GSTP1*C Val¹⁰⁴(104G), site)Val¹¹³(113T) Theta 22q11 GSTT1*null Absent Allele GSTT1 GSTT1*T1

[0037] Possible GSTM1 genotypes are GSTM1*A/GSTM1*A, GSTM1*A/GSTM1*B,GSTM1*/B/GSTM1*B, GSTM1*A/GSTM1*null, GSTM1*B/GSTM1*null, andGSTM1*null/GSTM1*null.

[0038] In the assay method of the instant invention, GSTM1 alleles canbe detected by fluorescent, allele-specific PCR using two forwardprimers, M1FA (SEQ ID NO: 1) and M1FB (SEQ ID NO: 2), and one reverseprimer, M1R (SEQ ID NO: 3). The sequences of these primers are asfollows: M1FA: 5′-(TET)TTGGGAAGGCGTCCAAGCAC-3′ M1FB:

M1R:

[0039] The polymorphic nucleotides in the primers are placed on the 3′side of the forward primers in order to increase sequence specificity ofPCR amplification. TET and FAM represent green and blue fluorescenttags, respectively. The boxed sequence of M1FB represents a non-sequencespecific tail added to create PCR product length differences between the142 base pair TET-tagged GSTM1*A PCR product and the 145 base pairFAM-tagged GSTM1*B PCR product. The boxed sequence of the reverse primerwas added to prevent spectral overlap between TET- and FAM-tagged PCRproducts. Both the length of the non-sequence specific tails and thetags may be easily varied within the scope of the instant invention.

[0040] CDK2 is co-amplified as a positive control forGSTM1*null/GSTM1*null genotypes and to determine GSTM1 gene dosage. CDK2forward and reverse primers are CDK2F (SEQ ID NO: 14) and CDK2R (SEQ IDNO: 15), respectively. Sequences of CDK2F and CDK2R are as follows:CDK2F: 5′-CCTATTCCCTGGAGATTCTG CDK2F: 5′-(FAM)AAACTTGGCTTGTAATCAGGC

[0041] To determine gene dosage of GSTM1*A or GSTM1*B by competitivePCR, peak areas of GSTM1*A or GSTM1*B, and CDK2 PCR products can bequantified by polyacrylamide gel electrophoresis and fluorescencedetection using an ABI PRISM® 373 Sequence Detection System (appliedBiosystems, Foster City, Calif.). Peak areas of GSTM1*A or GSTM1*Bproducts can them be compared to the peak area of the CDK2 PCR productin order to determine GSTM1 gene dosage, thereby differentiating betweenthe genotypes GSTM1*A/GSTM1*A and GSTM1*B/GSTM1*B and the genotypesGSTM1*A/GSTM1*null or GSTM1*B/GSTM1*null.

[0042] This step may be carried out under the following reaction orother suitable conditions: CDK2F 0.25 μM, M1R 0.5 μM, Taq PlatinumPolymerase 0.25 U (Life Technologies, Rockville, Md.), spermidine HCl0.2 mM, MgCl₂ 1.5 mM, NaCl 40 mM, Tris-HCl (pH 8.3) 10 mM, dNTPs 200 μMeach. Reaction volume is 20 μl. Samples are amplified in an MJResearchPTC250 Thermacycler (MJResearch, Inc., Watertown, Mass.) or othersuitable equipment. The amplification parameters can be as follows:denaturing for 5 minutes at 94 C, 25 temperature cycles (comprising 20seconds at 94 C, 20 seconds at 52 C, and 40 seconds at 72 C), followedby a final extension for 10 minutes at 72 C.

[0043] A next step in the assay method is assaying for the polymorphicalleles of GSTM3. This subgroup includes the GSTM3*A and GSTM3*B allelicvariants. These alleles are thought to result from a 3 base-pairdeletion in intron 6 which generates a YY1 negative transcription factorrecognition site in GSTM3*B that does not exist in GSTM3*A. Possiblegenotypes include GSTM3*A/GSTM3*A, GSTM3*A/GSTM3*B, and GSTM3*B/GSTM3*B.

[0044] In the assay of the instant invention, GSTM3 alleles may bedetected by fluorescent, allele-specific PCR using one forward primer,M3F (SEQ ID NO: 4) and one reverse primer, M3R (SEQ ID NO: 5). Thesequences for these primers are as follows: M3F:

M3R:

[0045] The three base pair deletion for GSTM3*B is located internal tothe primers M3F and M3R. The boxed sequences represent non-sequencespecific tails added to adjust PCR product length. Amplification ofGSTM3*A results in a 287 base pair PCR product.

[0046] This amplification step is compatible with, and thus may beperformed concurrently with, GSTT1 PCR amplification. One suitable setof reaction conditions follow: T1F 0.5 μM, T1R 0.5 μM, M3FA 0.5 EM, M3FB0.5 μM, M3R 0.5 μM, Taq Platinum Polymerase 0.25 U (Life Technologies,Rockville, Md.), spermidine HCl 0.2 mM, MgCl₂ 1.5 mM, NaCl 40 mM,Tris-HCl (pH 8.3) 10 mM, and dNTPs 200 μM each. Reaction volume is 20μl. Samples are amplified in an MJResearch PTC250 Thermacycler(MJResearch Inc., Watertown, Mass.) or other suitable equipment. Theamplification may occur under the following sample conditions:denaturing for 5 minutes at 94 C, 25 temperature cycles (20 seconds at94 C, 20 seconds at 58 C, 40 seconds at 72 C), followed by a finalextension for 10 minutes at 72 C. Other suitable amplification reactionsystems will be understood by one of skill in the art.

[0047] A third step is the detection of the polymorphic alleles ofGSTP1. These include GSTP1*A, GSTP1*B, GSTP1*C, and GSTP1*D. The GSTP1*Dallele has been observed, but only in very rare circumstances in twoindividuals. Watson et al., Carcinogenesis, 19:275-280 (1998). The GSTP1polymorphic alleles are thought to result from A to G and C to Ttransitions at nucleotides +313 of exon 5 and +341 of exon 6. Thesetransitions result in ATC (Ile¹⁰⁴) to GTC (Val¹⁰⁴) and GCG (Ala¹¹³) toGTG (Val¹¹³). Specifically, GSTP1*A codon 104 is ATC, coding for Ile¹⁰⁴,and codon 113 is GCG, for Ala¹¹³. GSTP1*B codon 104 is GTC, coding forVal¹⁰⁴, and codon 113 is GCG for Ala¹¹³. GSTP1*C codon 104 is GTC,coding for Val¹⁰⁴, and codon 113 is GTG for Val¹¹³. GSTP1*D codon 104 isATC, coding for Ile¹⁰⁴, and codon 113 is GTG for Val¹¹³. GSTP1 genotypesexpected to be common include GSTP1*A/GSTP1*A, GSTP1*A/GSTP1*B,GSTP1*A/GSTP1*C, GSTP1*B/GSTP1*B, GSTP1*B/GSTP1*C, GSTP1*C/GSTP1*C.

[0048] The assay of the instant invention detects polymorphisms at codon104 of GSTP1 by fluorescent, allele-specific PCR using two forwardprimers, P1-104FA (SEQ ID NO: 6) and P1-104FG (SEQ ID NO: 7), and onereverse primer, P1-104R (SEQ ID NO: 8). The sequences for these primersare as follows: P1-104FA:

P1-104FG:

PO1-104R:

[0049] As noted with GSTM1 above, the underlined polymorphic nucleotidesare placed at the 3′ side of the forward primers in order to increasesequence specificity of PCR amplification. The boxed sequences representnon-sequence specific tails added to adjust PCR product length and tocreate differences in length between PCR products containing an A incodon 104, (thus producing a 150 base pair PCR product) and productscontaining a G in codon 104 (thus producing a 153 base pair PCRproduct). Both the length of the non-sequence specific tails and thetags may be easily varied within the scope of the instant invention.

[0050] The reaction for this assay at codon 104 may proceed as followsor under other suitable reaction conditions: P1-104FA at 0.25 μM,P1-104FG 0.125 μM, P1-104R 0.5 μM, Taq Platinum Polymerase 0.25 U (LifeTechnologies, Rockville, Md.), spermidine HCl 0.2 mM, MgCl₂ 1.5 mM, NaCl40 mM, Tris-HCl (pH 8.3), 10 mM, dNTPs 200 μM each. The reaction volumeis 20 μl. Samples can be amplified in an MJResearch PTC250 Thermacycler(MJResearch Inc., Watertown, Mass.) or with other suitable equipment.The amplification may occur under the following conditions: denaturingfor 5 minutes at 94 C, 25 temperature cycles (20 seconds at 94 C, 20seconds at 64 C, 40 seconds at 72 C), followed by a final extension for10 minutes at 72 C.

[0051] Polymorphisms of codon 113 of GSTP1 are similarly detected byfluorescent, allele-specific PCR using two forward primers, P1-l 13FC(SEQ ID NO: 9) and P1-113FT (SEQ ID NO: 10), and one reverse primer,P1-113R (SEQ ID NO: 11). The sequences for these primers are as follows:P1-113FC:

P1-113FT: 5′-(FAM)GGTGTCTGGCAGGAGGC-3′ P1-113R:

[0052] The underlined polymorphic nucleotides are placed at the 3′ sideof the forward primers in order to increase sequence specificity of PCRamplification. The boxed sequences represent non-sequence specific tailsadded to adjust PCR product length and to create differences in lengthbetween PCR products containing a C in codon 113 (producing a 130 basepair PCR product), and those containing a T in codon 113 (producing a126 base pair PCR product). Both the length of the non-sequence specifictails and the tags may be easily varied within the scope of the instantinvention.

[0053] One suitable set of reaction conditions for this step are asfollows: P1-113FC 0.25 μM, P1-113FT 0.125 μM, P1-113R 0.5 μM, TaqPlatinum Polymerase 0.25 U (Life Technologies, Rockville, Md.),spermidine HCl 0.2 mM, MgCl₂ 1.5 mM, NaCl 40 mM, Tris-HCl (pH 8.3) 10mM, dNTPs 200 μM each. Reaction volume is 20 μl. Samples may beamplified in an MJResearch PTC250 Thermacycler (MJResearch Inc.,Watertown, Mass.) or other suitable equipment. The amplification mayoccur under the following reaction conditions: denaturing for 5 minutesat 94 C, 25 temperature cycles (20 seconds at 94 C, 20 seconds at 64 C,40 seconds at 72 C), followed by a final extension for 10 minutes at 72C.

[0054] A final step in the first portion of the assay of the instantinvention is to detect the polymorphic alleles of GSTT1, includingGSTT1*null and GSTT1. As with the other null allele noted above, theGSTT1*null allele produces no functional product, thus operating as arecessive allele. Possible genotypes include GSTT1*T1/GSTT1*T1,GSTT1*T1/GSTT1*null, and GSTT1*null/GSTT1*null.

[0055] GSTT1 alleles are detected by fluorescent, allele-specific PCRusing the forward primer T1F (SEQ ID NO: 12), and T1R (SEQ ID NO: 13).The sequences of these primers are as follows: T1F:5′-(FAM)TTCCTTACTGGTCCTCACATCTC-3′ T1R:

[0056] The boxed sequence of T1R represents a non-sequence specific tailadded to adjust PCR product length. GSTT1 is amplified with GSTM3 as apositive control for GSTT1*null/GSTT1*null genotypes and to determineGSTT1 gene dosage. GSTM3 forward and reverse primers were used asspecified above. Both the length of the non-sequence specific tails andthe tags may be easily varied within the scope of the instant invention.

[0057] To determine gene dosage of GSTT1 by competitive PCR, peak areasof GSTM3*A or GSTM3*B, and GSTT1 PCR products are quantified bypolyacrylamide gel electrophoresis and fluorescence detection using anABI PRISM® 373 Sequence Detection System (Applied Biosystems, FosterCity; CA). Peak areas of GSTM3*A or GSTM3*B products are compared to thepeak area of the GSTT1 PCR product in order to determine GSTT1 genedosage, thereby differentiating between the genotypes GSTT1*T1/GSTT1*T1and GSTT1*null/GSTT1*null.

[0058] Sample reaction conditions that can be used are as follows: T1F0.5 μM, T1R 0.5 μM, M3FA 0.5 μM, M3R 0.5 μM, Taq Platinum Polymerase0.25 U (Life Technologies, Rockville, Md.), spermidine HCl 0.2 mM, MgCl₂1.5 mM, NaCl 40 mM, Tris-HCl (pH 8.3) 10 mM, dNTPs 200 μM each. Reactionvolume is 20 μl. Samples are amplified in an MJResearch PTC250Thermacycler (MJResearch Inc., Watertown, Mass.) or other suitableequipment. A suitable set of reaction conditions for the PCTamplification are as follows: denaturing for 5 minutes at 94 C, 25temperature cycles (20 seconds at 94 C, 20 seconds at 58 C, 40 secondsat 72 C), followed by a final extension for 10 minutes at 72 C.

[0059] A final step of the instant assay is to combine the PCR productsof each of the PCR reactions above. Following this, the PCR reactantsmay then be loaded into a single lane on an ABI PRISM® 373 SequenceDetection System (Applied Biosystems, Foster City, Calif.). For thisstudy, interpretation was performed manually although automateddetermination of GST genotypes is possible with ABI PRISM® software.

[0060] Referring to FIG. 1, the results of a high throughput genotypingconducted according to the method of the present invention is shown. Therows 1-5 each represent DNA samples from different human cell lines. Thegenotype of each DNA sample was determined by the differential length ofPCT products (increasing from left to right). Alleles are representedfrom left to right in the following order: CDK2 (a control), GSTP1 113C,GSTP1 113T, GSTM1*B, GSTM1*A, GSTM1*null, GSTP1 104A, GSTP1 104G,GSTT1*non-null, GSTT1*NULL, GSTM3B, AND GSTM3*A. The GSTP1 genotypedesignations GSTP1*A, GSTP1*B, and GSTP1*C were determined by thecombination of GSTP1 113 AND GSTP1 104 genotypes as follows:GSTP1*A=113C/104A, GSTP1*B=113C/104G, and GSTP1*C=113T/104G. Gene dosagefor GSTM1 AND GSTT1 was determined by comparing the area under the curve(AUC) of their products to the AUC of co-amplified homozygous controlsCDK2 and GSTM3, respectively. AUC ratios are near linear, but mayrequire optimization with the first set of patient samples.

[0061] Specifically, the genotype results are shown for five separatecell-line-derived DNA samples. Each line represents four different,combined PCR reactions from a single DNA sample source. The genotypecorresponding to each sample is shown at the end of each line. The genedosage for GSTM1 was determined by examining the ratios of the peak areaunder the curve of GSTM1 vs. CDK2. The gene dosage for GSTT1 wasdetermined by examining the ratios of the peak area under the curve ofGSTT1 vs. GSTM3. Ratios greater than 1.0 were interpreted to representgenotypes GSTM1*non-null/GSTT1*null. Independent experiments verifyingGSTM1 and GSTT1 gene dosage by an independent long range PCR assay.

[0062] Though the data in FIG. 1 was gathered using DNA gathered fromtissue-culture cell lines, the assay method has been shown successful inassaying DNA derived from patient-derived peripheral leukocytes. It isfurther useful in analyzing DNA isolated from buccal epithelial cells(taken in some instances from mouthwash samples) as well as dried bloodspots taken from Guthrie cards. This characteristic of sample sourceversatility renders the assay method of the instant review highly usefulfor large clinical trials.

[0063] Finally, the design and cost of the high-throughput assay methodof the instant invention render it attractive to researchers. Morespecifically, the methodology of the instant invention allows thedetermination of polymorphic alleles of four different GST genes for 96patient samples within a period of about 10 hours at a current cost ofapproximately $7.50 per sample.

[0064] As a result of these characteristics of the instant invention, itis apparent that the method of the instant invention lends itself to awide array of applications. These include the pharmacogeneticapplications of: detecting individuals at risk for specific diseases inorder to aid in the development of prevention strategies, tailoringtherapeutic regimens for patients predicted to have decreasedtherapeutic response to medical therapy, tailoring therapeutic regimensfor patients predicted to have increased therapy-related toxicity, andallowing for the patient-specific utilization of allele-specificsmall-molecule inhibitors for the purpose of reversing chemotherapyresistance among cancers over-expressing certain GST polymorphicalleles.

[0065] The present invention may be embodied in other specific formswithout departing from its structures, methods, or other essentialcharacteristics as broadly described herein and claimed hereinafter. Thedescribed embodiments are to be considered in all respects only asillustrative, and not restrictive. The scope of the invention is,therefore, indicated by the appended claims, rather than by theforegoing description. All changes that come within the meaning andrange of equivalency of the claims that follow this specification are tobe embraced within their scope.

1 15 1 20 DNA Artificial Sequence Synthetic oligonucleotide. 1ttgggaaggc gtccaagcac 20 2 23 DNA Artificial Sequence Syntheticoligonucleotide. 2 tctttgggaa ggcgtccaag cag 23 3 31 DNA ArtificialSequence Synthetic oligonucleotide. 3 gtttcttctg cttcacgtgt tatgaaggtt c31 4 26 DNA Artificial Sequence Synthetic oligonucleotide. 4 gtttctcctcagtacttgga agagct 26 5 26 DNA Artificial Sequence Syntheticoligonucleotide. 5 gtttctcaca tgaaagcctt caggtt 26 6 25 DNA ArtificialSequence Synthetic oligonucleotide. 6 gtttctgacc tccgctgcaa ataca 25 728 DNA Artificial Sequence Synthetic oligonucleotide. 7 gtttctcttgacctccgctg caaatacg 28 8 23 DNA Artificial Sequence Syntheticoligonucleotide. 8 gtttctcagc ccaagccacc tga 23 9 21 DNA ArtificialSequence Synthetic oligonucleotide. 9 ctttggtgtc tggcaggagg t 21 10 17DNA Artificial Sequence Synthetic oligonucleotide. 10 ggtgtctggc aggaggc17 11 25 DNA Artificial Sequence Synthetic oligonucleotide. 11gtttcttggt ctcccacaat gaagg 25 12 23 DNA Artificial Sequence Syntheticoligonucleotide. 12 ttccttactg gtcctcacat ctc 23 13 27 DNA ArtificialSequence Synthetic oligonucleotide. 13 gtttctacag actggggatg gatggtt 2714 20 DNA Artificial Sequence Synthetic oligonucleotide. 14 cctattccctggagattctg 20 15 21 DNA Artificial Sequence Synthetic oligonucleotide.15 aaacttggct tgtaatcagg c 21

1. A method of detecting the presence of glutathione S-transferasealleles comprising the steps of: obtaining a sample of genomic DNA;amplifying a first portion of the sample with primers for GSTM1 andCDK2; amplifying a second portion of the sample with primers for GSTT1and GSTM3; amplifying a third portion of the sample with primers forGSTP1; and detecting the alleles and their gene dosage; wherein saiddetecting shows the presence or absence of the alleles of GSTM1, GSTM3,GSTT1, and GSTP1, and the gene dosages of GSTM1 and GSTT1.
 2. The methodof claim 1, wherein at least one primer further comprises a fluorescenttag selected from the group consisting of TET, FAM(6FAM), 5FAM, TAMRA,HEX, R110, JOE, RG6, NED, and ROX.
 3. The method of claim 2, wherein thePCR products of the amplification of the first, second, and thirdportions of the sample are combined prior to the step of detecting thepresence and the gene dosage of the alleles.
 4. The method of claim 3wherein the step of detecting the presence or absence of the alleles andtheir gene dosage comprises conducting a gel electrophoresis of thesample using a single lane.
 5. The method of claim 4, wherein the stepof detecting the presence or absence of the alleles and their genedosage further comprises fluorescence detection to determine thepresence of PCR products for the specific alleles.
 6. The method ofclaim 5, wherein the step of detecting the alleles and their gene dosagefurther comprises the steps of: comparing the areas under the peak ofGSTM1*A or GSTM1*B PCR products with the area under the peak of the CDK2PCR products to determine the gene dosage of GSTM1; and comparing theareas under the peak of GSTM3*A or GSTM3*B PCR products with the areaunder the peak of the GSTT1 PCR products to determine the gene dosage ofGSTT1.
 7. The method of claim 3, wherein the step of detecting thealleles and their gene dosage comprises capillary electrophoresis. 8.The method of claim 7, wherein the step of detecting the alleles andtheir gene dosage further comprises fluorescence detection to determinethe presence of PCR products for the specific alleles.
 9. The method ofclaim 8, wherein the step of detecting the alleles and their gene dosagefurther comprises the steps of: comparing the areas under the peak ofGSTM1*A or GSTM1*B PCR products with the area under the peak of the CDK2PCR products to determine the gene dosage of GSTM1; and comparing theareas under the peak of GSTM3*A or GSTM3*B PCR products with the areaunder the peak of the GSTT1 PCR products to determine the gene dosage ofGSTT1.
 10. The method of claim 2, wherein the primers further compriseinternally-biotinylated nucleotides.
 11. The method of claim 10, whereinthe step of detecting the presence and gene dosage of the allelescomprises the steps of: combining the PCR products of the amplificationof the first, second, and third portions of the sample; exposing themixture to streptavidin-coated magnetic or non-magnetic beads; anddetecting the presence or absence of allele-specific PCR productsthrough the use of a microplate reader capable of indicating thepresence or absence of allele-specific PCR products.
 12. The method ofclaim 2, wherein the steps of amplifying a first portion of the samplewith primers for GSTM1 and CDK2; amplifying a second portion of thesample with primers for GSTT1 and GSTM3; amplifying a third portion ofthe sample with primers for GSTP1; and detecting the presence and thegene dosage of the alleles are accomplished using real-time PCR, whereinamplification and detection of the alleles of GSTM1, GSTT1, GSTM3, andGSTP1 and the determination of the gene dosages of GSTM1 and GSTT1alleles is conducted simultaneously.
 13. The method of claim 1, whereinat least one of the primers comprises at least one radiolabelednucleotide.
 14. The method of claim 13, wherein the step of detectingthe alleles and their gene dosage comprises detection of theradiolabeled nucleotides.
 15. A high-throughput assay for the detectionof glutathione S-transferase polymorphic alleles comprising the stepsof: obtaining a sample of genomic DNA; conducting PCR amplification ofthe genomic DNA using primers for GSTM1, CDK2, GSTT1, GSTM3, and GSTP1;combining the PCR reaction products into one mixture; and detecting thepresence and the gene dosage of the alleles.
 16. The high-throughputassay of claim 15, wherein at least one of the primers further comprisesa label selected from the group consisting of: TET, FAM(6FAM), 5FAM,TAMRA, HEX, R110, JOE, RG6, NED, ROX, biotinylated deoxynucleotides,radioactive phosphorus deoxynucleotides, and radioactive sulfurphosporothioates.
 17. The high-throughput assay of claim 16, wherein thestep of detecting the presence and the gene dosage of the allelescomprises loading the mixture into a single gel lane and conducting gelelectrophoresis.
 18. The high-throughput assay of 16, wherein the stepof detecting the presence and the gene dosage of the alleles comprisescapillary electrophoresis.
 19. A method of identifying the alleles ofglutathione S-transferase present in a sample of genetic material,comprising the steps of: conducting the PCR amplification of the GSTM1alleles and CDK2 present in a first portion of the sample using primerscomprising polynucleotide sequences substantially identical to SEQ IDNO: 1, nucleotides 3-23 of SEQ ID NO: 2, nucleotides 9-31 of SEQ ID NO:3, SEQ ID NO: 14, and SEQ ID NO: 15; wherein at least one of saidprimers further comprises a signal marker; conducting the PCRamplification of the GSTT1 and GSTM3 alleles present in a second portionof the sample using primers comprising polynucleotide sequencessubstantially identical to nucleotides 7-26 of SEQ ID NO: 4, nucleotides7-26 of SEQ ID NO: 5, SEQ ID NO: 12, and nucleotides 6-27 of SEQ ID NO:13; wherein at least one of said primers further comprises a signalmarker; conducting the PCR amplification of the GSTP1 alleles present ina third portion of the sample using primers comprising polynucleotidesequences substantially identical to nucleotides 7-25 of SEQ ID NO: 6,nucleotides 10-28 of SEQ ID NO: 7, nucleotides 6-23 of SEQ ID NO: 8,nucleotides 4-21 of SEQ ID NO: 9, SEQ ID NO: 10, and nucleotides 7-25 ofSEQ ID NO: 11; wherein at least one of said primers further comprises asignal marker; and detecting the presence and gene dosage of the allelesamplified above; wherein said detecting shows the presence or absence ofthe alleles of GSTM1, GSTM3, GSTT1, and GSTP1, and the gene dosage ofGSTM1 and GSTT1.
 20. The method of claim 19, wherein the signal markersare selected from the group consisting of: TET, FAM(6FAM), 5FAM, TAMRA,HEX, R110, JOE, RG6, NED, ROX, biotinylated deoxynucleotides,radioactive phosphorus deoxynucleotides, and radioactive sulfurphosporothioates
 21. The method of claim 20, wherein the PCR products ofthe amplification steps are combined prior to the step of detecting thepresence of the alleles of GSTM1, GSTM3, GSTT1, and GSTP1, and the genedosage of GSTM1 and GSTT1.
 22. The method of claim 21 wherein the stepof detecting the presence of the alleles of GSTM1, GSTM3, GSTT1, andGSTP1, and the gene dosage of GSTM1 and GSTT1 comprises analyzing theallele makeup and gene dosage of the sample using a single lane of a gelelectrophoresis.
 23. The method of claim 21, wherein the step ofdetecting the presence of the alleles of GSTM1, GSTM3, GSTT1, and GSTP1,and the gene dosage of GSTM1 and GSTT1 comprises capillaryelectrophoresis.
 24. The method of claim 21, wherein the primers furthercomprise internally-biotinylated nucleotides.
 25. The method of claim24, wherein the step of detecting the presence and gene dosage of thealleles comprises the steps of: combining the PCR products of theamplification of the first, second, and third portions of the sample;exposing the mixture to streptavidin-coated magnetic or non-magneticbeads; and detecting the presence or absence of allele-specific PCRproducts through the use of a microplate reader capable of indicatingthe presence or absence of allele-specific PCR products.
 26. The methodof claim 19, wherein the steps of amplifying a first portion of thesample with primers for GSTM1 and CDK2; amplifying a second portion ofthe sample with primers for GSTT1 and GSTM3; amplifying a third portionof the sample with primers for GSTP1; and detecting the presence and thegene dosage of the alleles are accomplished using real-time PCR, whereinamplification and detection of the alleles of GSTM1, GSTT1, GSTM3, andGSTP1 and the determination of the gene dosage of GSTM1 and GSTT1alleles is conducted simultaneously.
 27. The method of claim 19, whereinat least one of the signal markers comprises at least one radiolabelednucleotide.
 28. The method of claim 27, wherein the step of detectingthe presence of the alleles of GSTM1, GSTM3, GSTT1, and GSTP1, and thegene dosage of GSTM1 and GSTT1 comprises detecting radiolabeled PCTproducts.
 29. A PCR primer comprising a nucleotide sequence selectedfrom the group of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO:
 13. 30. ThePCR primer of claim 29, said primer further comprising a signal markerselected from the group consisting of: TET, FAM(6FAM), 5FAM, TAMRA, HEX,R110, JOE, RG6, NED, ROX, biotinylated deoxynucleotides, radioactivephosphorus nucleotides, and radioactive sulfur phosporothioates.
 31. ThePCR primer of claim 29, wherein the primer further comprisedradiolabeled nucleotides.
 32. The PCR primer of claim 29, wherein theprimer further comprised internally-biotinylated nucleotides.